The present invention relates to the use of novel calibration standards or controls in the quantitative analysis of bilirubin in biological fluids by diazotization and direct reading methods.
Bilirubin, a principal component of bile pigment in a body fluid, is produced by the decomposition of heme from the hemoglobin in red blood cells. Two fractions of bilirubin are present in blood serum. One is named free or non-conjugated bilirubin and the other conjugated bilirubin since it has become conjugated with glucuronic acid and rendered water-soluble. Conjugated bilirubins react easily with diazonium ions and their quantity can be determined by colorometry. Thus, the conjugated bilirubins are commonly referred to as direct bilirubins.
Free bilirubin, on the other hand, is hydrophobic and difficult to quantitatively analyze directly by diazotization. Consequently, its quantity is typically ascertained indirectly by first determining the total bilirubin and the conjugated bilirubin and subtracting the latter from the former. Hence, the free or non-conjugated bilirubins are commonly referred to as indirect bilirubins. Measurement of the total bilirubin involves the use of solubilizing agents such as methanol, dimethylsulfoxide and benzoate-caffeine to dissolve the water-insoluble non-conjugated bilirubins and thereby insure dissolution of both bilirubin fractions for diazotization.
The content of bilirubin in blood increases in response to an increase in the decomposition of hemoglobin as well as a decrease in liver function. Accordingly, the quantitative analysis of both direct and indirect bilirubins is considered to be an indispensable clinical test item.
Now the purpose of a calibration standard or control in colorimetric measurements such as spectrophotometric measurements is to provide a standard agent from which an unknown can be measured. Generally, highly purified preparations of the analyte are used for this purpose. Unfortunately, purified bilirubin is a compound which is highly unstable to light and temperature. For this reason, it is necessary to store bilirubin in dark containers in the absence of air in a cold place. It has been demonstrated that bilirubin standards can deteriorate two percent per month even at -23.degree. C. (See Tritz, Fundamentals of Clinical Chemistry, W.B. Saunders Company, Philadelphia, Pa., 2nd Edition (1976), 1034-1043, and Dowmas et al, Clinical Chemistry, 19, (9):984 (1973).
There are several calibration controls commercially available. The most common is lyophilized serum which contains bilirubin. An example is LYPOCHEK, a trademarked product of Bio-Rad Company. Bilirubin in this control is stable for three days at 2.degree.-8.degree. C. after the addition of water. Lyophilized controls suffer from the inaccuracy of rehydration by the operator and by their very short life.
Another type of control commercially available is CHEMTRAK, a trademarked liquid bilirubin control/calibrator of Medical Analysis Systems, Inc. This control has the advantage of being a liquid control and having a shelf life of sixty days, if not opened. Once opened, however, the control is only stable for fourteen days at 2.degree.-8.degree. C. Moreover, the material is light sensitive. In the products described, bilirubin is not used as a control substance. Instead, derivatives of bilirubin are used rather than true bilirubin. The agents in CHEMTRAK.RTM. Liquid Bilirubin Control and Sta-Bil.RTM. L Liquid Bilirubin Control are bilirubin extracts and synthetic derivatives such as bilirubin ditaurate. All compounds which are analogues to bilirubin in structure have been found to be unstable.
Accordingly, it is evident that a need exists for an improved, more stable standard.